Scientific Reports (Mar 2023)

RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding

  • Jun Komatsu,
  • Alba Cico,
  • Raya Poncin,
  • Maël Le Bohec,
  • Jörg Morf,
  • Stanislav Lipin,
  • Antoine Graindorge,
  • Hélène Eckert,
  • Azadeh Saffarian,
  • Léa Cathaly,
  • Frédéric Guérin,
  • Sara Majello,
  • Damien Ulveling,
  • Anaïs Vayaboury,
  • Nicolas Fernandez,
  • Dilyana Dimitrova,
  • Xavier Bussell,
  • Yannick Fourne,
  • Pierre Chaumat,
  • Barbara André,
  • Elodie Baldivia,
  • Ulysse Godet,
  • Mathieu Guinin,
  • Vivien Moretto,
  • Joy Ismail,
  • Olivier Caille,
  • Natacha Roblot,
  • Carine Beaupère,
  • Alexandrine Liboz,
  • Ghislaine Guillemain,
  • Bertrand Blondeau,
  • Pierre Walrafen,
  • Stuart Edelstein

DOI
https://doi.org/10.1038/s41598-023-31915-y
Journal volume & issue
Vol. 13, no. 1
pp. 1 – 11

Abstract

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Abstract Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology optimized for samples of fresh cells. Complexes of one cell paired with one barcoded bead are stabilized by a chemical linker and dispersed in a hydrogel in the liquid state. Upon gelation on ice the complexes are immobilized and physically separated without requiring nanowells or droplets. Cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcoded beads for further processing with reverse transcription and preparation for cDNA sequencing. As a proof of concept, analysis of PBMC using RevGel-seq achieves results similar to microfluidic-based technologies when using the same original sample and the same data analysis software. In addition, a clinically relevant application of RevGel-seq is presented for pancreatic islet cells. Furthermore, characterizations carried out on cardiomyocytes demonstrate that the hydrogel technology readily accommodates very large cells. Standard analyses are in the 10,000-input cell range with the current gelation device, in order to satisfy common requirements for single-cell research. A convenient stopping point after two hours has been established by freezing at the cell lysis step, with full preservation of gene expression profiles. Overall, our results show that RevGel-seq represents an accessible and efficient instrument-free alternative, enabling flexibility in terms of experimental design and timing of sample processing, while providing broad coverage of cell types.